Bacteria such as Escherichia coli have developed various mechanisms to overcome toxic environments that are otherwise unfavorable for their survival. One important strategy that bacteria use to subvert toxic compounds, including heavy metal ions, is the expression of membrane transporters that recognize and actively export these toxic compounds out of bacterial cells, thereby allowing the bugs to survive in extremely toxic conditions. Our long-term goal is to elucidate the structures and fundamental mechanisms that give rise to heavy metal ion recognition and extrusion in heavy metal efflux proteins. The primary target of this proposal is the E. coli CusABC efflux system that recognizes and extrudes silver and copper ions out of the bacterial cell. CusA consists of 1,047 amino acid residues. It is an inner membrane transporter, which belongs to the resistance-nodulation-division (RND) protein superfamily. CusC is a 457 amino acid polypeptide that forms an outer membrane channel in E. coli. These two membrane proteins interact with each other, in conjunction with a membrane fusion protein CusB (379 amino acids), to mediate the extrusion of heavy metal ions across both membranes of E. coli. It has been proposed that CusB may act as an adaptor that brings CusA and CusC together to form the CusABC tripartite complex. This efflux complex makes direct contact with the metal ions and selectively expels them out of the cell. We recently cloned, expressed, and purified the full-length CusA, CusC, and CusB efflux proteins. We also crystallized each protein in detergent solution using vapor- diffusion. X-ray diffraction data were collected from cryocooled crystals at a synchrotron light source. The best CusA, CusC and CusB crystals diffracted to resolutions of 3.1, 3.6, and 2.8 E, respectively, with space groups determined to be R32, P21, and I222. The specific aims are to determine the structural basis of heavy-metal ion interactions with: (1) the CusA inner membrane efflux pump, (2) the CusC outer membrane channel, and (3) the CusB membrane fusion protein. PUBLIC HEALTH RELEVANCE: Heavy-metal resistance pathogens appear to be on the rise. This proposal deals with the structural basis of Ag+/Cu+ recognition and extrusion in transmembrane efflux transporters. Thus, the research will help to improve our knowledge of silver and copper resistance in pathogenic bacteria, and will provide a platform for thinking about novel metal-based antimicrobial therapeutic strategies that will lead to new treatments.